This study was conducted during the period of August 2021 to April 2022 and divided into two parts. The first part involved the isolation and characterization ofSalmonellafrom 200 diseased broiler chickens collected from farms in Dakahlia Governorate, Egypt, with the detection of its antimicrobial susceptibility. The second experimental part involved in ovo inoculation of probiotics and florfenicol to evaluate their effects on hatchability, embryonic viability, growth performance traits and the control of multidrug-resistantSalmonellaEnteritidis infections post hatching. The point prevalence ofSalmonellain the internal organs of diseased chickens was 13% (26/200), including 6 serotypes:S.Enteritidis, S.Typhimurium, S.Santiago, S.Colindale, S.Takoradiand S.Daula. Multidrug resistance was found in 92% (24/26) of the isolated strains with a multiantibiotic resistance index of 0.33–0.88 and 24 antibiotic resistance patterns. The in ovo inoculation of probiotic with florfenicol showed significant improvement in the growth performance parameters compared with other groups and had the ability to prevent colonization of multidrug resistantS.Enteritidis in the majority of the experimental chicks, and the remaining chicks showed very low colonization, as detected by RT‒PCR. These findings suggested the application of in ovo inoculation techniques with both probiotics and florfenicol as a promising tool to control multidrug-resistantS.Enteritidis in poultry farms.