This study was conducted to characterize and compare the protective effects of various innate immune stimulants against yolk sac infection (YSI) caused by an avian pathogenic Escherichia coli in young chicks. The immune stimulants were administered alone or in various combinations of unmethylated CpG oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (Poly I:C), and avian antimicrobial peptides (AMPs). Routes included in ovo or in ovo followed by a subcutaneous (S/C) injection. CpG alone and in combination with Poly I:C, truncated avian cathelicidin (CATH)-1(6-26), avian beta defensin (AvBD)1, and CATH-1(6-26) + AvBD1, were administered in ovo to 18-day-old embryonated eggs for gene expression and challenge studies. Next, CpG alone and the potentially effective formulation of CpG + Poly I:C, were administrated via the in ovo route using 40 embryonated eggs. At 1 day post-hatch, half of each group also received their respective treatments via the S/C route. Four hours later, all chicks were challenged using E. coli strain EC317 and mortalities were recorded for 14 d. The first challenge study revealed that amongst the single use and combinations of CpG with different innate immune stimulants, a higher protection and a lower clinical score were offered by the combination of CpG + Poly I:C. The second challenge study showed that this combination (CpG + Poly I:C) provides an even higher level of protection when a second dose is administered via the S/C route at 1 day post-hatch. The current research highlights the efficacy of a combination of CpG + Poly I:C administered either in ovo or in ovo along with a S/C injection and its potential use as an alternative to antibiotics against yolk sac infection in young chicks.