Se nanoparticles are attractive nanomaterials for application in medicine and therapeutics due to their antimicrobial and anticancer features. They are used as potential chemotherapeutic agents in a variety of cancers due to their broad range of biological activity, minimal toxicity, and prolonged effects. Therefore, the aim of this study was to optimize the biosynthesis of SeNPs using cell free extract of marine bacteria. The potent marine bacterial isolate was identified as Bacillus halotolerans based on 16 S rRNA gene sequencing. Characterization of SeNPs by UV–Vis spectroscopy, SEM–EDX, X-ray diffraction (XRD), and TEM techniques disclosed the biosynthesis of spherical shaped SeNPs with an average particle size of 7.50 - 19.17 nm. Consequently, SeNPs preparation using cell free extract of Bacillus halotolerans was statistically optimized using Plackett–Burman Design. Among five selected variables, reaction temperatures followed by the cell concentration were found to be highest significant factors in the biosynthesis of SeNPs. The acute and subacute toxicity studies were evaluated in mice to screen SeNPs safety. Biochemical, haematological and histopathological analysis were investigated. SeNPs showed no acute toxicity up to dose of 2000 mg/kg. In sub-acute toxicity, no remarkable differences between the control and SeNPs treated mice groups in terms of body weight, biochemical parameters, haematological indices or microscopic appearance of liver and kidneys were observed. Therefore, our results indicate that SeNPs could be used safely as anticancer agent without any toxic risk.