Psychobiotics are unique class of probiotics that potentially improve mental health via the production of neuroactive compounds such as gamma-aminobutyric acid (GABA). This study aimed to evaluate and improve GABA-producing lactic acid bacterial strains from fermented food origin to be used as potential psychobiotics. Hundred and ten presumptive lactic acid bacteria (LAB) were isolated from rumi cheese, mish cheese and pickled green olive and screened for GABA production using thin-layer chromatography (TLC). The survival rate of isolates to tolerate acidic conditions at pH 2.5 for 3 hours and bile salt resistance at 0.3% was evaluated. Three selected isolates were identified by 16S rRNA as Lacticaseibacillus rhamnosus MROMI.22, Lacticaseibacillus casei MGOLIV.25 and Lacticaseibacillus chiayiensis MGOLIV.28, and their GABA contents were determined by gas chromatography‒mass spectrometry (GC‒MS) to be 8.61 mg/ml, 9.02 mg/ml and 10.16 mg/ml, respectively. The safety prerequisite was confirmed by the absence of hemolytic activity. The proteolytic activity, cell surface hydrophobicity and auto-aggregation of selected isolates were also evaluated. The selected isolates showed also, variable antibacterial activity degree against nine pathogenic strains and different antibiotic sensitivity to nine common antibiotics. Response Surface Methodology (RSM) was used for culture media conditions optimization for GABA production from L. chiayiensis MGOLIV.28 that was found to produce 65.70 mg/ml increase of GABA production more than 6.46 fold, with a 72 h fermentation time, 33.5°C temperature, 4.5 pH and at 3% mono-sodium glutamate concentration. The free radical scavenging activity of L. rhamnosus MROMI.22, L. casei MGOLIV.25 and L. chiayiensis MGOLIV.28 was 69.7ug/ml, 23.67ug/ml and 15.78ug/ml respectively. Also, they showed various cytotoxic activities degree.