Hematoxylin (HT) is a natural staining dye used in histopathology, often combined with Eosin for H&E staining. A poly(hematoxylin-co-l-lysine) (p(HT-co-l)) nanonetwork was synthesized through a one-step Mannich condensation reaction using formaldehyde as a linking agent. The resulting p(HT-co-l) nanogels had an average size of about 200 nm and exhibited a smooth surface and desirable functional groups such as -OH, -NH2, and -COOH, as recognized by FT-IR analysis. The isoelectric point (IEP) of the p(HT-co-l) nanogel was determined as pH 7.9, close to physiological environments, despite HT being acidic IEP at pH 1.7 and l-lysine being basic IPE at pH 8.7. The time-dependent swelling studies of p(HT-co-l) nanogels were carried out using dynamic light scattering (DLS) in different salt solutions, e.g., MgCl2, KNO3, KCl, PBS, and DI water environments revealed that nanogels have high swelling ability depending on the medium, e.g., >10-fold in a saline solution compared to distilled water within 1.5 h. Hydrolytic degradation studies in PBS demonstrated a linear release profile up to 125 h at 37.5 °C. The p(HT-co-l) nanogels also demonstrated significant antimicrobial and antifungal activities against E. coli (ATCC 8739), S. aureus (ATCC 6538), and C. albicans (ATCC 10231). Furthermore, biocompatibility tests indicated that p(HT-co-l) nanogels are more biocompatible than HT alone, as tested with human Nuli-1 bronchial epithelial cells.