The escalating resistance of bacteria to multiple antibiotics utilized over many years has prompted the search for innovative antibacterial agents possessing structural attributes enabling them to overcome the resistance mechanisms. To find a new antimicrobial substance, two pyrido-dipyrimidines (A and B) are tested against ten microorganisms using the microdilution method. The key structural difference between A and B lies in their functional groups located at the meta position, A has a hydroxyl (OH) group, while B contains a methoxy group. Tested compounds possessed significant antimicrobial activity. Among tested compounds, A showed stronger antibacterial activity (MIC values ranged from 0.31 to 0.62 mg/mL) and marked antifungal activity (MIC values ranged from 0.62 to 5 mg/mL). In silico research was in good agreement with the experimental results and showed that both molecules effectively inhibit the DNA gyrases of Gram (−) bacteria, in contrast to the Novobiocin molecule. It was also shown that both considered molecules show higher inhibitory activity against the corresponding hydrolase A. niger compared to the Miconazole molecule. While the inhibitory activity of molecules A and B against oxidoreductase C. albicans is lower, it closely competes with the inhibitory activity of the Miconazole. The better antimicrobial efficacy of molecule A is supported by its previously observed strong antioxidant potential, indicating its dual activity capability.