The sanitising effect of low concentration neutralised electrolysed water (LCNEW, pH: 7.0, free available chlorine (FAC): 4 mg/L) combined with ultrasound (37 kHz, 80 W) on food contact surface was evaluated. Stainless steel coupon was chosen as attachment surface for Escherichia coli ATCC 25922, Pichia pastoris GS115 and Aureobasidium pullulans 2012, representing bacteria, yeast and mold, respectively. The results showed that although LCNEW itself could effectively reduce survival population of E. coli ATCC 25922, P. pastoris GS115 and low concentration A. pullulans 2012 in planktonic status, LCNEW combined with ultrasound showed more sanitising efficacy for air-dried cells on coupons, with swift drops: 2.2 and 3.1 log CFU/coupon reductions within 0.2 min for E. coli ATCC 25922 and P. pastoris GS115, respectively and 1.0 log CFU/coupon reductions within 0.1 min for A. pullulans 2012. Air-dried cells after treatment were studied by atomic force microscopy (AFM)/optical microscopy (OM) and protein leakage analyses further. All three strains showed visible cell damage after LCNEW and LCNEW combined with ultrasound treatment and 1.41 and 1.73 μg/mL of protein leakage were observed for E. coli ATCC 25922 and P. pastoris GS115, respectively after 3 min combination treatment, while 6.22 μg/mL of protein leakage for A. pullulans 2012 after 2 min combination treatment. For biofilms, LCNEW combined with ultrasound also significantly reduced the survival cells both on coupons and in suspension for all three strains. The results suggest that LCNEW combined with ultrasound is a promising approach to sanitise food equipment.