The effects of snake venoms have been well known since ancient times. They contain a variety of biologically active proteins which have therapeutic potential. This study investigated the cytotoxic and antimicrobial activities of Anatolian Macrovipera lebetina obtusa venom against various cancer cells, Gram-negative and Gram-positive bacteria, and a fungal species. A549, HeLa, CaCo-2, U-87 MG and MCF-7 cancer cell lines and a normal cell line (Vero) were screened by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The antimicrobial activity was evaluated by determining the minimum inhibitory concentration (MIC) using the broth dilution method. The species included were Escherichia coli ATCC 25922, E. coli 0157:H7, Enterococcus faecalis, Enterococcus faecium DSM 13590, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Salmonella typhimurium CCM 5445, Proteus vulgaris ATCC 6957, Bacillus cereus ATCC 7064 and Candida albicans ATCC 10239. Half-maximal inhibitory concentration (IC50) values of M. l. obtusa venom on cultured cells varied from 1.18 +/- 0.11 to 12.80 +/- 0.22 mu g/ml, with the most potent activities against Vero, U-87 MG, MCF-7 and CaCo-2 cells. Venom showed moderate antifungal activity against C. albicans, with an MIC of 62.50 mu g/ml. In short, the venom of Anatolian M. l. obtusa showed promising results as a potential source of alternative therapeutics, cytotoxic and antifungal agent prototypes.