Some new quinolone condensed s‐triazine derivatives endowed with different heterocycles and 4‐aminobenzonitrile moiety has been synthesized and examined for their bioactivities against eight bacteria (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, and Shigella flexneri), two fungi (Aspergillus niger, Candida albicans) by using agar streak dilution method, and Mycobacterium tuberculosis H37Rv by using Lowenstein and Jensen MIC method. Upon preliminary biological screening, it was observed that the majority of the compounds were found to possess a significant broad spectrum antimicrobial (MICs: 6.25–25 µg/mL) and antitubercular (MIC: 12.5 µg/mL) potential. Hence, anti‐HIV activity against two types of HIV viral strains [HIV‐1 (IIIB) and HIV‐2 (ROD)] has been carried out using the MTT assay. From this bioassay, we have identified some potent inhibitors acting as anti‐HIV‐1 agents (IC50: 4.45 µg/mL) with promising therapeutic index of 16 for analogue 7 h. The structural assignments of the new products were carried out on the basis of IR, 1H NMR, 13C NMR spectroscopy, and elemental analysis.