Prodigiosin is a natural red pigment, it is an alkaloid secondary metabolite with a unique tripyrrole structure produced by Serratia marcescens, it's motile, short-rod shaped, G^- bacteria, classified in the large family of Enterobacteriaceae. 13 isolates were isolated from 32 soil samples, which showed clear red pigmented colonies. The Egyptian Serratia marcescens isolates were identified based on morphological, biochemical and then only the two best isolates for pigment production (M1, S1) were identified by MALDI-TOF mass spectrometry technique. The two selected species gave a score value between 2.565 to 2.668 (100%) were correctly identified by MALDI-TOF- MS to the genus and species levels. The average daily rate of pigment production by M1 and S1 isolates estimated by 421.6 and 326.1 unit/cell, respectively. Antibacterial activity of prodigiosin was determined by disc diffusion assay against 7 types of G^- and 4 types of G⁺ pathogenic bacteria. Minimum inhibitory concentration (MIC) and the concentration inducing 50% inhibition of the bacterial growth (IC₅₀)against the mentioned bacteria were determined and prodigiosin was proved to be effective in inhibiting bacterial growth. Prodigiosin possess antioxidant activity assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction method and this activity increased gradually with increasing concentrations.