The increase in food waste accumulation needs innovative valorization strategies that not only reduce environmental impacts but also provide functional applications. This study investigates the potential of almond hulls, an abundant agricultural by-product, as a source of bioactive compounds. For the first time, almond hull extract (AHE), was evaluated in terms of anti-adhesive and anti-biofilm activity against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 9637. The extract was obtained by an optimized eco-friendly green technique using ultrasound-assisted extraction (UAE), and it was characterized for its main compounds by high-performance liquid chromatography–mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis. Antimicrobial activity was evaluated on planktonic cells by minimum inhibitory/bactericidal concentration (MIC/MBC) and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Afterward, AHE activity was evaluated against the bacterial sessile phase, both against in-formation and mature biofilm. Finally, the toxicity of the extract was tested on normal human adult cells (HDFa) by an MTT test. The principal active compounds present in AHE belong to the polyphenol group, in particular, the phenolic acid (Hydroxycinnammic sub-class) and, more significantly, the flavonoid class. The results showed that the extract has a relevant antimicrobial activity against the planktonic cells of both tested strains. Moreover, it significantly inhibited bacterial adhesion and promoted biofilm removal, highlighting its potential as a sustainable antimicrobial agent. The MTT test on human fibroblasts showed that the extract is not toxic for normal human cells. This research highlights how food waste valorization could have a high potential in the antimicrobial field.