Salmonellosis is a commonly occurring bacterial disease in chickens that leads to increased mortality, as well as a decline in the quality and quantity of hen eggs. This, in turn, causes financial losses in the poultry industry. The objective of this research was to investigate the potential benefits of using Lactobacillus plantarum and novel Bacillus salmalaya 139SI as probiotics in the drinking water of newly hatched chicks. The study aimed to evaluate the growth performance of laying hens and the quality of eggs produced during a later stage of development. The supplementation of L. plantarum resulted in the highest increase in both chicken body weight and egg weight, followed by the treatment with B. salmalaya 139SI. The different components of the eggs, such as the eggshells, egg yolks, and egg whites, were examined to detect the presence of Salmonella spp. Various tests were conducted, including biochemical testing, serotyping, conventional detection using specific agars, and a modern detection method utilizing 16S rRNA sequencing. The results from all the tests indicated that both probiotics, L. plantarum and B. salmalaya 139SI, can produce eggs free from Salmonella contamination. Moreover, the treatment with L. plantarum demonstrated significant protective effects against Salmonella infection in the eggs, surpassing the protective capacity of B. salmalaya 139SI. As conventional testing methods for Salmonella identification are not entirely reliable, the utilization of modern sequencing tools proved to be more accurate and dependable in detecting the infection in eggs.