Lactic acid bacteria (LAB) play pivotal roles in food fermentation and preservation, contributing to the flavour and texture of various food products. This study aimed to isolate LAB from diverse food sources and characterise them using 16s rRNA to species levels, and to develop cost-effective and sustainable media for LAB to promote their growth as alternatives to conventionally used laboratory media, and finally enhance LAB’s application in fermentation industry. Chicken meat and fresh produce samples were collected from retailers in Brisbane, Australia. Isolation and identification were conducted using conventional plating, followed by molecular identification with 16s rRNA sequencing. Physiological characterisations of LAB were performed to determine growth and metabolism in DeMan Rogosa and Sharpe (MRS) agar and broth. Additionally, fermentation assays were conducted by inoculating 20 LAB isolates in 11 different kinds of culture media formulation to evaluate their acid production ability. A total of 518 isolates were gained from raw chicken meat, fresh produce, and vegetables by plating at 22 °C (n = 274) and 30 °C (n = 244), using 16s rRNA sequencing, a wide range of LAB were identified across various food sources. The study categorised these isolates into mesophilic and psychrotrophic groups according to different plating temperatures (22 °C and 30 °C), revealing significant diversity with common genera such as Lactobacillus, Enterococcus, Lactococcus, and Leuconostoc. Media trials with 11 simplified formulations using 20 LAB isolates representing 8 species, led to good growth and lower fermentation pH values. A broth composed of 2% glucose + 1% yeast extract + 1% peptone + 1% tryptone led to 0.2-0.45 lower final pH units for Lactobacillus sakei and Lactobacillus graminis than in normal MRS broth. A simple broth composed of glucose 1.33% + yeast extract 1.33% + potassium phosphate 0.27% led to a lower final pH 0.3 units for Leuconostoc kimchii compared to MRS broth. In conclusion, this study contributes to our understanding of LAB diversity, culture media development and optimisation, and underscores LAB’s potential applications in the food industry, laying a foundation for further research and innovation in food microbiology and biotechnology.