The Spaniard's preparations for Wimbledon were dealt a big blow as the veteran Belgian recorded a 7-6 6-3 victory.
The 33-year-old former Wimbledon semi-finalist took the opener 7-3 in the tie-break after there were no breaks of serve.
The first break of the match came in the eighth game of the second set as defending champion Ferrer dropped his serve to love and Malisse, now ranked 60th in the world, completed the job in 88 minutes.
Ferrer was riding high following his first Grand Slam final appearance at Roland Garros and followed fourth seed Benoit Paire out of the tournament.
It takes the tally of seeds eliminated to six after John Isner, Marcos Baghdatis, Victor Hanescu and Daniel Gimeno-Traver crashed out on Monday.
Second seed Stanislas Wawrinka survived though as he defeated Steve Darcis 7-6(5) 6-4.
ATP s'Hertogenbosch Round 1 results
2-Stanislas Wawrinka (Switzerland) beat Steve Darcis (Belgium) 7-6(5) 6-4
Jan Hernych (Czech Republic) beat David Goffin (Belgium) 6-3 3-6 7-6(4)
Nicolas Mahut (France) beat Ricardas Berankis (Lithuania) 6-1 6-4
Xavier Malisse (Belgium) beat 1-David Ferrer (Spain) 7-6(3) 6-3
Michael Llodra (France) beat 4-Benoit Paire (France) 3-0 (Paire retired)
5-Jeremy Chardy (France) beat Lucas Pouille (France) 7-5 6-4
Marius Copil (Romania) beat Rogerio Dutra Silva (Brazil) 6-7(4) 6-4 6-2
Lu Yen-Hsun (Taiwan) beat Edouard Roger-Vasselin (France) 6-1 7-6(3)