Edible packaging films have received a considerable attention in recent years because of their advantage over the synthetic films in the reduction of environmental pollution. The aim of the research was development of optimal edible packaging bio-films from gelatin and glycerol by varying the concentration of (5,10 and 15 wt/wt) and (4,8,12 and 16v/wt) respectively with the main responses of physical property (solubility, swelling and WVTR) and mechanical property (tensile strength and elongation at break), and re-preparing a packaging bio-film by adding Nigella Sativa seed extract together with gelatin and glycerol optimal response results and finding its antimicrobial activities of the developed packaging bio-films against food-born pathogens. The factorial response optimizer results were gelatin concentration (10g gelatin/100ml aqueous solution) and glycerol concentration (4ml glycerol/100ml aqueous solution), solubility (54.76%), swelling (43.99%), WVTR (0.145gm/day cm2), tensile strength (20.302Mpa), and elongation at break (14.95%). The optimal re-prepared packaging bio-films with the addition of Nigella Sativa seed extract with the concentration (12.5%, 25% and 50%) were also analyzed its Antimicrobial activities against targeted food-born pathogens (Escherchia coli, Salmonella typhi, Staphylococcus aureus, and Pseudomonas aeruginosa). The Nigella Sativa seed extract showed result (30, 23 and 19.5mm inhibition zone on Escherchia coli , 30, 25.5 and 20mm inhibition zone on Salmonella typhi and 30, 24 and 20 inhibition zone on Staphylococcus aureus with the concentration of Nigella Sativa seeds extract of 75, 50 and 25% for the mentioned pathogens respectively) except for (Pseudomonas aeruginosa) whereas the bio-film that contain the Nigella Sativa seed extract showed only surface inhibition.