The leaves of Psidium guajava L. are an agro-industrial by-product with an outstanding content of polyphenolic compounds; however, there are many factors which can affect the phytochemical profile when valuing this type of plant material, such as temperatures and extraction times involving in the extraction methods applied. In this context, this study analyzed the impact of different extraction methods (Soxhlet, maceration and ultrasound-assisted extraction) on the phytochemical profile (FTIR and UPLC-MS) and the antioxidant activity (ABTS, FRAP and Folin–Ciocalteu) of guava leaf extracts. A yield of phenolic compounds per gram of guava leaf was obtained within the range of 16 to 45 mg/g; on the other hand, the IC50 values determined with the ABTS assay ranged between 78 ± 4 to 152 ± 12 µg/mL. The methanolic extract obtained by Soxhlet was the one with the best reducing power, both in the FRAP assay and in the Folin–Ciocalteu assay. Finally, bioactive compounds such as quercetin, kaempferol and avicularin were identified in the guava leaf extract. It was concluded that the purification of polyphenolics compounds improves the antioxidant capacity, and that the extraction method greatly influences the phytochemical profile and activity of the extracts.