Crude Tetrodotoxin (TTX) extracts of liver, skin and muscles which isolated from Puffer fish, Lagocephalus sceleratus (L. sceleratus) were tested for their antimicrobial activity besides TTX levels (µg/g) using LC–MS–MS (Liquid Chromatograph / mass spectrometry) in cold and warm season and TTX identification by HPLC (High Performance Liquid Chromatograph), TLC (Thin Layer Chromatograph) and UV spectrum (Ultra Violet spectrophotometer). The results obtained showed interesting antibacterial and antifungal activity against Crude extracts of TTX isolated from liver, skin and muscles of L. sceleratus. The crude TTX of liver extract showed quite promising broad-spectrum antibacterial activity due to its capacity to inhibit the growth of almost bacterial tested strains. It has shown the maximum activity against Escherichia coli as 22.24 ± 0.96 in gram negative bacteria and Bacillus subtilis as 17.51 ± 0.51 in gram positive bacteria and minimum activity against Vibiro cholerae as 9.22 ± 0.65. The maximum antifungal activity was observed to crude TTX of liver extract against Aspergillus fumigatus as 19.21 ± 0.59 and minimum activity against Trichophyton rubrum as 5.33 ± 0.52 in crude TTX of skin extract. Changes in the levels of TTX in the livers, skins and muscles as a function of season and sex were determined. The highest TTX levels in livers, skins and muscles of female fish were 30.33, 3.38 and 2.26 µg/g, respectively in winter. The TTX level in the muscle of male fish in summer was 0.66µg/g but was otherwise below the toxic limit. Consequently, it can be dangerous to consume puffer fish, including the edible muscle, from Mediterranean Sea. The present investigations have helped to focus on some bioactive toxins isolated from marine resources; these molecules, which possess antimicrobial activities, could be used for human and TTX levels in relationship to season and sex of L. sceleratus in the Mediterranean Sea, Egypt.