In the post-antibiotics era, prebiotics are proposed as alternatives to antibiotic growth promoters in poultry production. The goal of this study was to compare in ovo method of prebiotic delivery with in-water supplementation and with both methods combined (in ovo+in-water) in broiler chickens. Two trials were conducted. Trial 1 was carried out to optimize the doses of two prebiotics, DN (DiNovo®, extract of beta-glucans) and BI (Bi2tos, trans-galactooligosaccharides), for in ovo delivery. The estimated parameters were hatchability and bacteriological status of the newly hatched chicks. Prebiotics were dissolved in 0.2 ml of physiological saline, at the doses: 0.18, 0.88, 3.5 and 7.0 mg/embryo; control group (C) was injected in ovo with 0.2 ml of physiological saline. Trial 2 was conducted to evaluate effects of different prebiotics (DN, BI and raffinose family oligosaccharides (RFO)) delivered in ovo, in-water and in a combined way (in ovo+in-water) on broiler chickens performance. The results of the Trial 1 indicated that the optimal dose of DN and BI prebiotics delivered in ovo, that did not reduce chicks’ hatchability, was 0.88 mg/embryo (DN) and 3.5 mg/embryo (BI). Both prebiotics numerically increased number of lactobacilli and bifidobacteria in chicken feces (P>0.05). In Trial 2, all prebiotics (DN, BI and RFO) significantly increased BW gain compared with the C group (P<0.05), especially during the first 21 days of life. However, feed intake and feed conversion ratio were increased upon prebiotics delivery irrespective of method used. Injection of prebiotics in ovo combined with in-water supplementation did not express synergistic effects on broilers performance compared with in ovo injection only. Taken together, those results confirm that single in ovo prebiotics injection into the chicken embryo can successfully replace prolonged in-water supplementation post hatching.