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The antimicrobial assessments of two new series of nicotinonitriles and pyrido[2,3‐d]pyrimidines were performed using amoxicillin and nystatin as reference standards. Outstanding antifungal activities were achieved by some target compounds; for instance, compounds 7 and 9 displayed a minimal inhibitory concentration (MIC) value of 1.95 µg/ml toward Candida albicans, compound 11 showed a potent anti‐Rhizopus effect (MIC 1.95 µg/ml) and compound 14 elicited remarkable antifungal effects against both Aspergillis niger and C. albicans (MIC 1.95 µg/ml). However, pyrido[2,3‐d]pyrimidines 12, 14, and 16 showed moderate antibacterial activities against some gram‐negative bacteria. The antibiofilm results of these compounds against resistant strains of Proteus mirabilis were better than those of Pseudomonas aeruginosa. Docking studies of these hits at the DNA gyrase active site revealed affinity and …