Innovative nanodrug are in high demand for the novel antimicrobial agents development that have Sustained release dosage in gastrointestinal simulation. Therefore, this study looked at the effect of mixing selenium nanoparticles (SeNPs) and their composites with cefepime, embedded in polyvinyl alcohol (PVA) with polyhydroxybutyrate (PHB) matrices for controlled drug release to overcome pathogenic bacteria. These results showed that cefepime (Cef) released more slowly at pH 2 than at pH 7.4. Zero-order, first-order, Higuchi, Hixon–Crowell, and Korsmeyer–Peppas equations were used to study the drug release mechanism at various pH values (2, 7.4). According to zeta potential analysis, the composite exhibited good stability, indicating to strong structure. The nanocomposite possesses an encouraging antimicrobial potential against pathogenic strains S. aureus, E. coli, and P. aeruginosa, Kb. pneumonia strain. Moreover, SeNPs and PHB/PVA/SeNPs/Cef nanocomposites recorded excellent antioxidant activity (87.35% and 79.9%). The total phenolic compounds for SeNPs and PHB/PVA/SeNPs/Cef nanocomposite are 875 mg gallic acid/g and 1160 mg gallic acid/g in 200 µg/mL of SeNPs that activated their antioxidant properties. MTT analysis was showed that PHB/PVA/SeNPs/Cef has no cytotoxic effect on normal HEK- 293 cells and RPE-1 normal cells at even 50 µg/mL of SeNPs for 24 h. Moreover, the coagulation cascade was unaffected by the nanocomposites at the high concentration. Also, there was no evidence of haemolysis when the nanocomposite interacted with the constituents. The release test exhibited controlled drug release of Cef from composite, indicating its potential for use in drug delivery systems.