In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η 6-p-cymene)Ru(nitrophen)Cl]Cl; C 22 H 21 Cl 2 N 3 O 2 Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemi-diaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC 50) of enzyme activity ranging from 16 to 26 μM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC 50 = 19.44 μM), without affecting the directly-evoked muscle single twitch up to 40 μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neos-tigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration dependent manner (IC 50 = 7.6 μM) without any significant change in the resting membrane potential of muscle fibers up to 40 μM. Finally, C1-Cl (5-40 μM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 μM and (ii) their frequency at 10 μM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-de-polarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.