Probiotics can improve broiler performance and reduce pathogens. Because the hatchery can be a source of contamination, delivering probiotics to the embryo before hatch is desirable. To date, probiotics have primarily been injected into eggs manually. Therefore, the objective of this study was to deliver various probiotic bacteria into broiler hatching eggs using an automated commercial in ovo injection system to evaluate hatchability of fertile eggs (HF). Three separate experiments were conducted using Lactobacillus acidophilus, Bacillus subtilis, or Bifidobacterium animalis. In each experiment, 7 treatments (non-injected control; dry punch control; diluent-injected control; and injections of 103 cfu, 104 cfu, 105 cfu, or 106 cfu of bacteria/50 μL of diluent) were evaluated using 10 replicates per treatment. For each experiment, 2,490 eggs were obtained from a commercial hatchery. Eggs were incubated under standard incubation conditions. At 10 d of incubation (doi), eggs were candled, and infertile eggs were removed. On 18 doi, all eggs were injected with the appropriate treatment using an automated in ovo injection system. Once all eggs were injected, they were transferred to hatching baskets and placed into the hatcher. On 21 doi, chicks were removed from the hatcher, counted, and weighed. Hatch residue analysis was conducted to determine infertile, early dead, mid dead, late dead, pipped, cracked, contaminated, and cull chick statuses of all unhatched eggs. Injecting L. acidophilus, even at a concentration as high as 106 cfu/50 μL, did not impact hatch residue analysis (P > 0.05). However, HF was significantly less for eggs treated with B. subtilis than for control eggs (P < 0.0001). For the non-injected control, HF was 91%, but as concentration of B. subtilis increased, HF decreased to as low as 1.67% for the 105 cfu treatment. Late deads, pipped, and contaminated egg percentages were higher, and chick BW was lower for B. subtilis treatment groups compared to controls. In conclusion, L. acidophilus and B. animalis but not B. subtilis, appear to be suitable candidates for in ovo injection as probiotics.