The utilization of medicinal plants and their drugs have the advantage of reducing side effects compared with manufactured antimicrobials. Artificial drugs have unpleasant side effects, further, the number of drug resistant pathogens is increasing, thus huge challenge for control of resistant pathogens. Therefore, the current research explores the potential of partially purified bioactive compounds from Dodonaea viscosa against certain human pathogens. Healthy leaves of D. viscosa (L.) were collected, extracted and optimized with different solvents. Preliminary phytochemical screening of the extracts was done and antibacterial activities were tested against human pathogens. The active crude extract was further purified by column chromatography and the homogeneity was confirmed by thin layer chromatography (TLC). The partially purified compounds were screened further for antibacterial, antibiofilm and anticancer activities. The crude ethanol extract of D. viscosa leaves showed the presence of phytochemical like tannins, alkaloids, flavanoids, terpenoids, glycosides, steroids and phenols. Ethanol extract exhibited the maximum zone of inhibition (11 mm) against S. agalactiae, B. cereus, S. typhi and E. coli at 15 mg when compared with other bacteria. Column chromatography fractions Dv12 and Dv20 exhibited the maximum zone of inhibition against B. cereus. 1000 μg of Dv12 partially purified compound against streptococcus isolates in glass test tube showed biofilm inhibition range of 34.4%-63.1%. Whereas B. cereus, S. aureus, S. typhi, and K. pneumoniae showed 31.1%-53.6% biofilm inhibition compared to curcumin control. Active fractions of Dv12 and Dv20 increased concentration confirmed that the gradual decrease in cell density and possesses growth inhibition towards A 549 human lung adenocarcinoma cells. We have extracted the bioactive compounds from D. viscosa (L.) leaves and tested the activity of a partially purified compound against human pathogenic bacteria, biofilm formation and cytotoxicity against A 549 human lung adenocarcinoma cells. The purified bioactive compounds might be used as therapeutic agents against different microbial infections such as skin infection, throat infection and other infectious diseases.