Pereskia aculeataMiller, is an unconventional food plant native to South America. This study aimed to investigate the influence of different ultrasonic extraction times (10, 20, 30, and 40 min) on the phytochemical profile, antioxidant and antibacterial activities of ethanolic extracts obtained from lyophilizedPereskia aculeateMiller (ora-pro-nobis) leaves, an under-researched plant. Morphological structure and chemical group evaluations were also conducted for the lyophilizedP. aculeateleaves. The different extraction times resulted in distinct phenolic content and Antioxidant Activity (ATT) values. Different extraction time conditions resulted in phenolic compound contents ranging from 2.07 to 2.60 mg EAG.g−1of extract and different ATT values. The ATT evaluated by DPPH was significantly higher (from 61.20 to 70.20 μM of TE.g−1of extract) in extraction times of 30 and 40 min, respectively. For ABTS, it varied between 6.38 and 10.24 μM of TE.g−1of extract and 24.34 and 32.12 μM ferrous sulp.g−1of extract. All of the obtained extracts inhibited the growth ofStaphylococcus aureus, particularly the treatment employing 20 min of extraction at the highest dilution (1.56 mg.mL−1). Although liquid chromatography analyses showed that chlorogenic acid was the primary compound detected for all extracts, Paper Spray Mass Spectrometry (PS-MS) suggested the extracts contained 53 substances, such as organic, fatty, and phenolic acids, sugars, flavonoids, terpenes, phytosterols, and other components. The PS-MS proved to be a valuable technique to obtain theP. aculeateleaves extract chemical profile. It was observed that the freeze-drying process enhanced the conservation of morphological structures ofP. aculeateleaves, as evidenced by scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FTIR) identified carboxyl functional groups and proteins between the 1000 and 1500 cm−1bands in theP. aculeateleaves, thus favoring water interaction and contributing to gel formation. To the best of our knowledge, this is the first study to evaluate different times (10, 20, 30 and 40 min) for ultrasound extraction ofP. aculeateleaves. The polyphenols improved extraction, and high antioxidant activity demonstrates the potential for applyingP. aculeateleaves and their extract as functional ingredients or additives in the food and pharmaceutical industries.