The present work aimed to optimize amino acids production by Bacillus amyloliquefaciens 35s from chicken feather using response surface methodology (RSM) and applying the produced amino acids in biological control of powdery mildew disease. Variables affecting amino acids production from B. amyloliquefaciens 35s were screened using Plackett Burman design. Factors showed to be significant to produce amino acids were optimized using central composite design (CCD) of RSM at three coded levels (-1, 0, +1). All obtained data were analyzed by ANOVA with post hoc multiple comparison analysis performed using Tukey’s HSD. TGY (tryptone, glucose, yeast extract) was the basal medium. Impacts of medium components were studied using Plackett-Burman design. “Design Expert 12” StateEase was used to analyze the experimental Plackett- Burman design. Feather, yeast extract, CaCO3, KH2PO4 and agitation rate (using shake flask) were optimized statistically by the CCD design of the RSM. Validation of statistical model of the medium components was done by carrying out the experiment at optimum conditions of the process parameters as determined by the model. Amino acids types were identified by amino acid analyzer. Among the significant medium components, feather, yeast extract, CaCO3, KH2PO4 and agitation rate showed to have significant effect on amino acids production. Predicted maximum amino acids production was observed near the mid-point concentrations of the mentioned above factors and the experimental value (6.55 g/l) was very close to the predicted value of (6.60 g/l) predicted by the model. Amino acid analyzer analysis for the produced amino acids shows the release of asparagine, threonine, serine, glutamine, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenyl alanine, histidine, lysine and arginine, respectively. Effect of chicken feather hydrolysate on cucumber powdery mildew disease was tested by applying foliar application with different concentrations (0, 5, 10, 15, and 20% v/v), then degree of infection and sporulation on cucumber plants were assessed. The lowest concentration of chicken feather hydrolysate showed the lowest effect on disease index. Conversely, cucumber resistance increased by the increasing of feather’s hydrolysate concentrations associated with a clear reduction in the disease index and sporulation. Aside from, extra foliar application showed a decrease in the disease index for all concentrations compared with untreated plants. Foliar application with 20% concentration of chicken feather hydrolysate had the highest stimulation of the studied oxidative enzymes i.e., peroxidase (POX), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and superoxide dismutase (SOD).