The champions trail their rivals by four points heading into the Clasico at the Bernabeu, with Atletico Madrid sandwiched between the two sides three points behind Carlo Ancelotti's league leaders.
With just 10 fixtures remaining in the domestic season, Martino feels it is now or never for Barcelona.
"This is probably our last opportunity to keep ourselves in the fight for the title," Martino said on Saturday.
"After this game there will be nine (matches to play), and it's not the same as closing up on a one point gap than four or seven is.
"At this stage, teams - mainly the ones playing for the title - do not normally make mistakes."
Martino identified Real's consistency as the defining factor in their rise to the top of the table, and not any difference in quality between the top two in the Spanish top flight.
"Both sides face this Clasico knowing perfectly well what they have to do," he said.
"The only difference up until now is that Real Madrid have been consistent since the last Clasico and we haven't.
"The quality of both teams is clear. The thing is we haven't been as consistent as them so that made us drop points and therefore drop down the table. But this is not a matter of quality at all."
Martino also said he had no qualms about letting Brazilian starlet Neymar know how good he was, ahead of Barca's visit to the Bernabeu.
"Neymar was great against Manchester City, and secondly I will reaffirm what I've said plenty of times - he's amongst the five best footballers in the world," he said.
"I don't need one game or half of it, not even 10 (minutes), to believe his qualities. So within my powers, I'll try to make him not think that he's just one more player in the squad because he's not."
