Broiler chickens, because of the intensive production systems in which they are bred and their high metabolic demands, are generally vulnerable to infectious diseases and thus, immune support is an essential element of sustainable poultry production. Traditional prophylactic methods using antibiotics and synthetic immune boosters raise concerns regarding antimicrobial resistance and environmental impact. Consequently, plant-based natural immunomodulators represent a considerably safer and more environmentally sustainable option. Thus, the oyster mushroom (Pleurotus ostreatus)—a mushroom widely attested for its bioactive compounds with immunostimulatory properties—was thus evaluated for its ability to enhance the humoral immunity of broilers through in ovo administration. A total of 300 hatching eggs were incubated, out of which 165 were fertile (55% fertility rate). On the 18th day of incubation, the fertile eggs were divided into three groups for injection (55 eggs each): a control (without injection), 0.1 ml/egg extract, and 0.2 ml/egg extract. The hatched chicks were reared for 8 weeks under a deep litter system and were given feed and water ad libitum. The birds' immune status was evaluated by immunization with sheep red blood cells (SRBC) on day 29 post-hatch, and the antibody titres were measured by the haemagglutination assay. Results showed that broilers from eggs injected with 0.2 ml extract were found to have a significantly higher (p<0.05) humoral immune response (mean titre of 5.96) than those of the control and 0.1 ml groups. In summary, in ovo injection of not more than 0.2 ml of water extract of Pleurotus ostreatus can be a method to activate the humoral immune system of broiler chickens and, therefore, can be regarded as a safe and effective nutritional approach for the contemporary poultry sector.