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Eight <i>N</i>-[2-(2′,3′,4′-tri-<i>O</i>-acetyl-α/β-<span style="font-variant: small-caps;">d</span>-xylopyranosyloxy)ethyl]ammonium bromides, a new class of <span style="font-variant: small-caps;">d</span>-xylopyranosides containing a quaternary ammonium aglycone, were obtained. Their complete structure was confirmed using NMR spectroscopy (¹H, ¹³C, COSY and HSQC) and high-resolution mass spectrometry (HRMS). An antimicrobial activity against fungi (<i>Candida albicans</i>, <i>Candida glabrata</i>) and bacteria (<i>Staphylococcus aureus</i>, <i>Escherichia coli</i>) and a mutagenic Ames test with <i>Salmonella typhimurium</i> TA 98 strain were performed for the obtained compounds. The greatest activity against the tested microorganisms was shown by glycosides with the longest (octyl) hydrocarbon chain in ammonium salt. None of the tested compounds exhibited mutagenic activity in the Ames test.