Novel η4-acyloxy-cyclohexadiene-Fe(CO)3 complexes (with variable length of the acyloxy chain) were synthesized as potential enzyme-triggered carbon monoxide (CO)-releasing molecules (ET-CORMs). The molecular structure of two complexes was additionally confirmed by X-ray crystallography. The enzyme-triggered CO-releasing activity of the compounds was assessed under physiological conditions (37 °C, 0.1 M phosphate buffer, pH = 7.4) by headspace gas chromatography (GC) and additionally by means of a myoglobin assay (UV). The relative rate of CO release and the amount of liberated CO were found to depend on the length of the acyloxy chain and its position at the diene unit (outer or inner position). Some of the new ET-CORMs exhibited very good biological activity as assessed in different cellular assays (cytotoxicity, protective effect against hypothermia-associated cell damage, and inhibition of TNF-α-mediated VCAM-1 expression).