In the present study, we use knipholone as a prototype molecule to identify new anti-infective agents. Since knipholone is insoluble in water, which would have a detrimental effect on its bioavailability and efficacy, we synthesized knipholone Mannich base derivatives (2–4) that have better predicted solubility and investigated their in vitro antimicrobial activity against eight pathogenic bacterial and fungal strains. The chemical structures of compounds 1–4 were elucidated from their 1H and 13C NMR data, and their antimicrobial activity evaluation was carried out by a broth microdilution MTT assay. Compound 3 exhibited the strongest efficacy against Staphylococcus epidermidis, with MIC value of 9.7 µg/mL. While 4 exhibited the best activity against Staphylococcus aureus, with an MIC value of 19.5 µg/mL, and was the only one to significantly inhibit the fungus Trichophyton mentagrophytes (MIC = 78.2 µg/mL). The study provides evidence for the antibacterial activity of aminoalkyl derivatives of knipholone.