The current study aimed to explore the phytochemical composition of guava (Psidium guajava) leaves extract and to assess its antimicrobial activity. This was done by in vitro assays, such as agar well diffusion assay and minimum inhibitory concentration (MIC) and in vivo antimicrobial testing in broiler chickens experimentally infected with Salmonella gallinarum (SG). Phytochemical screening showed the presence of flavonoids, tannins, terpenoids, phenols, and saponins. The zone of inhibition (ZOI) of guava leaves extract against SG according to the agar well diffusion assay was concentration-dependent. MIC was found to be 6.25 mg/mL. In the case of the in vivo study, 150 day-old broiler chicks were obtained and separated into five groups (A, B, C, D, and E). Group A was the control group that was not infected. Group B (preventive) was treated with the extract on days 4, 5 and 6 and then inoculated with SG (3×108 CFU/mL) on day 7. Group C: SG was infected but untreated. On day 7, group D was inoculated with SG and once clinical symptoms were experienced, the extract of guava leaves was used on days 11, 12 and 13. On day 7, Group E was inoculated and on days 11, 12 and 13 the standard antibiotic ciprofloxacin was administered. Hematological and serum biochemical evaluations on days 7, 14, and 21 demonstrated that guava leaves extract exerted a potent antimicrobial effect against Salmonella gallinarum, with efficacy comparable to the standard antibiotic ciprofloxacin in the in vivo model. All treated groups had their severity of gross and histopathological changes reduced by guava leaves extract. It is interesting to note that the conventional antibiotic ciprofloxacin was less effective in in vitro and in vivo analyses. The conclusion was that guava leaves extract is a strong antimicrobial agent against Salmonella gallinarum in broiler chickens.