Plant parasitic nematodes (PPN) cause significant losses in global agricultural production. The use of nematicides to prevent nematodes poses serious risks to both human health and the environment. Therefore, it is essential to find a safer alternative treatment. In this study, we explained the use of microbial bioagents such as alkaline protease to control Meloidogyne incognita. The isolate of lab418 was employed as a potential source of alkaline protease. By analyzing the sequence of the 16S rRNA gene, we identified the strain lab418 as Bacillus safensis lab418. This sequence has been assigned the accession number OR888822 in the NCBI database. To improve the production of alkaline protease by B. safensis, we employed the BOX-Behnken Design (BBD) and Plackett-Burman Design (PBD). Through optimization, we were able to significantly increase alkaline protease production to 245 U/mL, which represents a 1.92-fold improvement compared to the non-optimized production. This increase in alkaline protease production led to a mortality rate of 98% for M. incognita. Furthermore, positive effects on plant growth, specifically in terms of fresh weight and shoot and root length were recorded. These improvements can be attributed to the activation of biochemical characteristics related to plant defense mechanisms against nematodes, such as polyphenol oxidase, chitinase, glucanase, and phenolic compounds. Additionally, in silico analyses to investigate the anticipated protein interactions between alkaline protease and collagen present in the nematode cuticle was conducted, demonstrating the enzyme's binding and catalytic activity.