MicroRNAs (miRNAs) are short non-coding RNAs with important biological functions. Their presence in food and their potential role as new bioactive molecules is generating great expectations. For the first time, the miRNA fraction within food waste and algae has been studied, highlighting their bioactivity and its potential upcycling. MiRNAs from barley malt rootles, brewer’s spent grains, Spirulina, Chlorella, orange peels, pomegranate and apricot seeds, and watermelon waste were extracted using two kits, one with chloroform and a more sustainable alternative. Higher extraction yields were obtained with the chloroform-containing kit. Purity of extracts was confirmed by UV spectroscopy and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection. Further analysis by RP-HPLC and high-resolution mass spectrometry (MS) demonstrated the potential and feasibility of this technique for the analysis of miRNA although there are still some limitations to overcome before its routine application. Apricot seed miRNAs demonstrated remarkable anti-inflammatory and antioxidant capacity.