Two trials were conducted. The aim of the first trial, performed under field conditions, was the comparison of the rearing performance between the chickens injected in ovo with 1.9 mg of raffinose family oligosaccharides (RFOs) (experimental group E), and chickens administered with the antibiotics (control group C). Environmental and feed conditions were similar for both groups. The mortality, feed intake, and slaughter body weight were recorded from farm documentation. Feed conversion ratio (FCR) and European production efficiency factor (EPEF) were calculated. The aim of the second trial, performed under laboratory condition, was to determine the prebiotic effect of different doses of RFOs (0.5%, 1.0%, 3.0%, or 5.0%) on the growth of Bifidobacterium bifidum and Lactobacillus acidophilus in vitro. Prebiotic effect of RFOs administered with a single in ovo injection had a similar effect on broiler performance (survivability, growing period, and EPEF), to antibiotics given as growth promoters. Body weight and FCR was significantly higher in the group E in comparison to the group C. The prebiotic in vitro effect of RFOs was evident. The obtained data suggest that at least in the case of Bifidobacterium bifidum, RFOs could show the dose-dependent effects on the bacteria number. This work opens the opportunity of using, in technical scale, in ovo technology to deliver other bioactives to stimulate the prebiotic effect.