Cannabis (Cannabis sativa L.) for medical purposes has been legalized again in many countries in recent years. Currently, only two major cannabinoids (Δ9-THC and CBD) are considered in the legislation and medication, which is not sufficient in case of dried plant material or resulting extract. Other substances (mainly terpenes/terpenoids), or their specific combinations, could influence the resulting therapeutic effect for specific oncology diagnosis and specific patients. Six different genotypes (Conspiracy Kush, Jilly Bean, Jack Cleaner 2, Jack Skellington, Nordle and Nurse Jackie) were cultivated indoor at the Czech University of Life Sciences Prague. Ethanol extracts taken from the inflorescences were assayed for their content of main cannabinoids and terpenes/terpenoids. The extracts were used for in vitro cytotoxicity studies on hepatocarcinoma human cell lines Hep-G2 and colorectal carcinoma human cell lines Caco-2 and Ht-29. Healthy lung fibroblast MRC-5 and healthy intestinal cells FHs 74 Int were used to compare selectivity of cytotoxicity. The average content of Δ9-THC in extracts was 59.1 ± 2.43%, and of CBD 1.84 ± 0.17%. The content of main cannabinoids in the Nurse Jackie genotype extract was significantly greater than that of the other genotypes. Overall, more than 60 different terpenes/terpenoids were identified in the extracts. The major terpenes/terpenoids detected in most genotypes were limonene, linalool, α-terpineol, β-caryophyllene, trans-α-bergamotene, α-humulene, β-caryophyllene oxide, guaiol, γ-eudesmol, β-eudesmol and α-bisabolol. Differences in the terpene composition of individual genotypes were caused by minor terpenoids, such as β-ocimene, isopulegol acetate, β-elemene, β-selinene and spathulenol. All extracts were highly cytotoxic to Ht-29 colorectal carcinoma cells and showed positive selectivity compared to healthy FHs 74 Int colon cells. The Jack Cleaner 2 extract was cytotoxic to all cell lines tested at the lowest concentrations (8.48 ± 2.4-16.14 ± 0,07 μg/mL), but was positively selective only for colorectal cancer cells, especially Ht-29 and to a lesser extent for Caco-2. Similarly, the Nordle extract showed positive selectivity for Ht-29 and Caco-2 only. Jilly Bean was unique in this study, in that its extract functioned on all cell lines at the highest concentrations (20.13 ± 3.05-49.88 ± 1.5 μg/mL), whilst also being highly positively selective in all carcinoma lines (Ht-29, Caco-2 and Hep-G2 hepatocarcinoma) compared to healthy cell lines (FHs 74 Int and MRC-5). The results suggest that Δ9-THC and CBD are responsible for the in vitro cytotoxicity of the extracts, but observed differences in selectivity reveal their synergies with other substances. According to chemical analysis, higher concentrations of myrcene, β-elemene, β-selinene and α-bisabolol oxide found in the Jilly Bean genotype may positively affect the selectivity of cytotoxic activity. It is therefore vital that similar studies are performed on other cell lines, in order to be able to recommend these cannabis genotypes for preclinical and clinical studies, which are still lacking.