The work is included the preparation of two new benzylpenicillin derivatives by replacement alkyl groups (ethyl, propyl) instead of the acidic proton of the acylamino group (O=C-NH-), which attached to the position (6) of the benzylpenicillin molecule. The prepared derivatives are indicated by some physical measurements namely melting points, Thin layer chromatography technique and elemental analysis, besides the spectroscopic identification, UV, IR, and C13NMR. This work help us to obtain new derivatives with high biological activity against thirteen standard and isolated strains of Gram positive and Gram negative bacteria determined the antibacterial activity of the benzylpenicillin derivatives represented by inhibition diameter (IZ mm), minimum inhibitory concentration (MIC), media lethal dose(LD50) and cytotoxicity. The data of inhibition zones, and minimum inhibitory concentrations appeared that the biological activity anti-the test micro-organisms of the prepared derivatives (EBP, PBP), is more in comparison with biological activity of standard antibiotic (BP). Also the study was shown that the products with toxic effect for the human red blood cells, which reduced from 250 ppm of BP to 300 ppm for the products (EBP, PBP).