L-asparaginase (L-ASNase) functions as a chemotherapeutic enzyme with antitumor properties. It facilitates the degradation of L-asparagine (L-ASN), a vital amino acid required for the proliferation of tumor cells. In this study, we have isolated 177 L-ASNase-producing strains from the aquatic environment of the Arabian–Persian Gulf. The most potent isolate, ASP-J1-4, was an endophyte recovered from the seablite Suaeda maritima and was molecularly identified as B. xiamenensis (accession number PQ593941). The enzyme purified through DEAE-Sepharose displayed a molecular weight of 37 kDa based on the SDS-PAGE profile and lacked detectable L-glutaminase (L-GTNase) activity. Optimal enzyme activity was at 40 °C and pH 9.0, with stability at pH 7–9. The maximum stimulation effect was found in the presence of Fe³⁺, Mn²⁺, and Na⁺ ions, respectively. The enzyme demonstrated a V_max of 35.71 U/mL and a K_m of 0.15 mM. Interestingly, ASP-J1-4 L-ASNase showed a dose-dependent inhibition against human colon carcinoma (HCT-116) and cervical Henrietta Lacks (HeLa) cell lines, with IC₅₀ values of 15.42 µg/mL and 12.13 µg/mL, respectively. These findings collectively suggest a biocompatible, efficient, and robust enzyme for potential applications in tumor therapy after validation of in vivo studies and clinical trials. This study introduces the first deep screening program for L-ASNase-producing bacteria harboring in the Arabian–Persian Gulf region. In addition, it launches B. xiamenensis and other species as new sources of L-ASNase.