Studies highlight the cost-effectiveness and lower adverse effects of herbal remedies than allopathic medicines, making them attractive amidst growing microbial resistance concerns. This investigation explored the in vitro antiseptic properties of Phyllanthus niruri alcoholic extract (EA) against Escherichia coli, Staphylococcus aureus, Streptococcus spp., and Candida albicans. Various in vitro methods were employed, including disk diffusion, Minimum Inhibitory Concentration (MIC), and Bactericidal/Fungicidal Concentration (MBC/MFC). A skin cream formulation using P. niruri was developed, undergoing in vivo trials on ten feline subjects post-ovariosalpingohysterectomy (OSH). Felines were treated with P. niruri skin cream (100mg/ml), with 0.1% aqueous chlorhexidine as the positive control. Skin assessments included hyperemia, crust, edema, and exudation. Inhibition was observed in vitro at various EA concentrations, and cytotoxicity analysis showed no harm. The P. niruri skin cream at 100mg/ml exhibited consistent outcomes, with a minor deviation from the fifth day onwards, without compromising therapeutic efficacy (Tukey test, p < 0.05). The formulation demonstrated stable physical-chemical properties with no adverse feline reactions. These findings suggest the skin cream's in vivo antimicrobial potential as a future antiseptic alternative.